Glucose provides an essential nutrient source that supports glycolysis and the hexosamine biosynthesis pathway (HBP) to maintain tumour cell growth and survival. Here we investigated if short-term glucose deprivation specifically modulates the phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB) cell survival pathway. Insulin-stimulated PKB activation was strongly abrogated in the absence of extracellular glucose as a consequence of the loss of insulin-stimulated PI3K activation and short-term glucose deprivation inhibited subsequent tumour cell growth. Loss of insulin-stimulated PKB signalling and cell growth was rescued by extracellular glucosamine and increased flux through the HBP. Disruption of O-GlcNAc transferase activity, a terminal step in the HBP, implicated O-GlcNAcylation in PKB signalling and cell growth. Glycogenolysis is known to support cell survival during glucose deprivation, and in A549 lung cancer cells its inhibition attenuates PKB activation which is rescued by increased flux through the HBP. Our studies show that rerouting of glycolytic metabolites to the HBP under glucose-restricted conditions maintains PI3K/PKB signalling enabling cell survival and proliferation.